The rapid spread of antibiotic resistance in pathogenic bacteria has prompted a continuing search for new agents capable of antibacterial activity. Indeed, microbiologists today warn of a “medical disaster” which could lead back to the era before penicillin, when even seemingly small infections were potentially lethal. Thus, research into the design of new antibiotics is of high priority (1-3). One way to delay the emergence of antibiotic-resistance is to develop new synthetic materials that can selectively inhibit bacterial enzymes, via novel mechanisms of action. However, this approach is both time-consuming and financially prohibitive, yet remains indispensable if an acceptable level of care is to be provided in the immediate future. On the other hand, it may be less costly in time and money to employ strategies to circumvent existing bacterial resistance mechanisms and thereby to restore usefulness to antibacterials that have become compromised by resistance (4). The remarkable advances in recent years in elucidating the mechanisms of resistance to various clinical antibiotics on the molecular level provide complementary tools to this approach via structure-based and mechanism-based design.
One example of an important group of antibiotics which could benefit from such a redesign is the aminoglycoside class of antibiotics. Aminoglycosides (as shown in prior art FIG. 1) are highly potent, broad-spectrum antibiotics with many desirable properties for the treatment of life-threatening infections (5). Their history begins in 1944 with streptomycin and was thereafter marked by the successive introduction of a series of milestone compounds (neomycin, kanamycin, gentamycin, tobramycin, and others), which definitively established the usefulness of this class of antibiotics for the treatment of gram-negative bacillary infections (6). It is believed that aminoglycosides exert their therapeutic effect by interfering with translational fidelity during protein synthesis via interaction with the A-site rRNA on the 16S domain of the ribosome (7,8). Recent achievements in ribosome structure determination have provided fascinating new insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code.
Unfortunately, prolonged clinical use of currently available aminoglycosides has resulted in effective selection of resistance to this family of antibacterial agents (9). Presently, resistance to these agents is widespread among pathogens worldwide which severely limits their usefulness. The primary mechanism for resistance to aminoglycosides is the bacterial acquisition of enzymes which modify this family of antibiotics by acetyltransferase (AAC), adenyltransferase (ANT), and phosphotransferase (APH) activities (as shown in prior art FIG. 2). Among these enzyme families, aminoglycoside 3′-phosphotransferases [APH(3′)s], of which seven isozymes are known, are widely represented. These enzymes catalyze transfer of γ-phosphoryl group of ATP to the 3′-hydroxyl of many aminoglycosides, rendering them inactive because the resulted phosphorylated antibiotics no longer bind to the bacterial ribosome with high affinity. Due to the unusually broad spectrum of aminoglycosides that can be detoxified by APH(3′) enzymes, much effort has been put into understanding the structural basis for their promiscuity in substrate recognition and catalysis (10).
To tackle the problem of antibiotic resistance, many structural analogs of natural aminoglycosides have been synthesized over the past decade (11). In the majority of these studies a minimal structural motif, which is common for a series of structurally related aminoglycosides, has been identified and used as a scaffold for the construction of diverse analogs as potential new antibiotics (12). Some of the designed structures showed considerable antibacterial activities. Since the structural and mechanistic information on the target(s) of aminoglycosides and their respective resistance enzymes has only began to emerge in the past few years, this information stimulated novel developments in the de novo design of molecules that bind to the ribosomal target site and simultaneously are poor substrates for resistance-causing enzymes (13, 14). These results and design principles hold the promise of the generation of a large series of designer antibiotics uncompromised by the existing mechanisms of resistance.
In view of recent events, one particular disease for which effective therapeutic strategies are urgently required is anthrax.
Anthrax is an infectious disease caused by toxigenic strains of the Gram-positive Bacillus anthracis (15). If inhaled, B. anthracis spores rapidly reach the regional lymphonodes of the lungs where they germinate and release anthrax toxins (16). These toxins inhibit the adaptive immune response, thereby enabling the bacteria to reach the blood system where they cause bacteraemia and toxaemia, which rapidly kills the host. Non-toxigenic strains of B. anthracis are poorly pathogenic indicating that the anthrax toxins play a major role from the very beginning of infection to death. Since anthrax is asymptomatic until the bacterium reach the blood (15, 16), the development of anti-toxin therapeutics for preventive use or in combination with antibiotics, is of high urgency (17). Alternatively, and even preferably, the development of bifunctional substances that would inactivate the released toxins and in addition would function as an antibiotic would be highly beneficial.
The anthrax toxins consist of three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF) (18). Being individually nontoxic, their toxic effects during anthrax infection require cooperation: PA binds to a cell surface receptor and forms an oligomeric pore that translocates both EF and LF into the cytosol of target cells. Once inside the cell, EF causes edema via Ca2+/calmodulin-dependent adenylate cyclase activity. LF is a zinc-dependent endopeptidase that specifically cleaves most isoforms of mitogen-activated protein kinase kinases, thereby inhibiting one or more signaling pathways of the host macrophage (19). Through a mechanism that is not yet well understood, this results in the death of the host. Strains of B. anthracis deficient in EF remain pathogenic, while those lacking LF become attenuated. LF is therefore considered the dominant virulence factor of anthrax (20). Consequently, an intensive search for specific inhibitors of LF has been performed during the last years (17, 21-22).
The prior art does not teach or suggest a highly effective group of aminoglycosides which both share certain structural features of currently available aminoglycosides while also being able to reduce or eliminate antibiotic resistance. The prior art also does not teach or suggest such aminoglycosides which have reduced side effects. The prior art also does not teach or suggest such aminoglycosides which are capable of functioning both by inhibition of anthrax lethal factor, and as an antibiotic and are therefore highly effective for treatment of anthrax.
There is thus a widely recognized need for, and it would be highly advantageous to have, bifunctional antibiotics which both inactivate toxins and function as an antibiotic, while reducing or eliminating antibiotic resistance, and further resulting in reduced side effects. Such antibiotics would be highly beneficial in the treatment of bacterial infections such as anthrax.